Histomorphic Analysis of UV-C Radiation on Osseointegration of Titanium Implants in the Rabbits

Statement of the Problem: Unsuccessful implant integration leads to pain and implant mobility. Implant photo-functionalization by ultraviolet (UV) light has been suggested as a method that may stimulate osseointegration. Purpose: This study was conducted to analyze the histopathological feature of the titanium implant surface upon treatment with UV-C wave. Materials and Method: In this interventional study, twenty rabbits were enrolled. In the treatment groups, the titanium implants, irradiated earlier with UV-C for four hours laterally, were inserted in one of the femur bones. In the control group, the titanium implants without irradiation were inserted in the other femur bone of the rabbits. After two and four weeks, the animals were sacrificed, and then the samples were histologically and histo-morphometrically analyzed. In addition, the amounts of new bone formation, bleeding, and inflammation were recorded, and the data were subjected to statistical analysis. Results: The results confirmed that UV-C irradiation to titanium implants significantly improved new bone formation (p< 0.001). However, no significant new bone formation was observed between two and four weeks after implant insertion (p< 0.098). Conclusion: The study results showed that irradiating titanium implants with UV-C for four hours significantly improves osseointegration and new bone formation but does not considerably affect inflammation or bleeding around the implant. The study suggests that UV-C radiation can increase the success rate of implant treatment.


Introduction
The dental implant has become a routine clinical practice to replace a missing tooth. It helps patients feel more comfortable and functional than conventional prostheses [1][2][3]. Osseointegration is a practical and direct connection between the implant and live bone required for successful implant integration [2,4]. Unsuccessful implant integration leads to pain, implant mobility, and mastication force impairment [5][6]. Techniques such as sandblasting are widely used to increase surface roughness and improve osseointegration [7].
However, roughed surfaces are strongly associated with plaque accumulation [8]. Meanwhile, implant photo-functionalization by UV light has been suggested as an effective method to stimulate osseointegration [9][10]. The UV photo-functionalization was discovered in 1977 and defined as a change in titanium surface upon UV treatment. The treatment changes the hydrophobic features of the titanium surface into super-hydrophilic and enhances its biological capabilities [9][10][11][12]. UV photo functionalization is a phenomenon of surface modification by exposure to ultraviolet rays that alters the sur-face's physicochemical features and improves its biological capabilities [13]. Photo functionalization of titanium implants by UV is discovered as a simple and effective tool for osseointegration [10,[14][15]. This discovery shows that the physicochemical property of the titanium surface changes and transforms the hydrophobic property of the titanium surface into a solid hydrophilic feature. The phenomena are practically applied in microbiology and improve several biological capabilities [11][12]16].
Moreover, evidence suggests that photo-functionalization may improve osseointegration in the initial healing period [17]. Furthermore, the evidence indicates that photofunctionalization by UV may improve attachments, retention, and a functional cascade of osteogenic cells [13]. It is noteworthy that UV photo-functionalization is novel, simple, and low cost. However, further studies should validate these findings [13]. The potential of surface modification to make a successful dental implant material is highly associated with success in vitro and clinical studies. Therefore, a deep understanding of tissue response and osseointegration by dental implants is required [18]. Hence, this study was conducted to analyze the histopathological feature of the titanium implant surface upon treatment with UV-C wave.

Animals
The study protocol is illustrated in Figure 1. The experiment was conducted according to the guideline care of laboratory animals by the Tehran University of Medical Sciences. The study complied with the ARRIVE guidelines and National institutional guidelines for the care and use of laboratory animals. In this study, twenty healthy Albino rabbits were enrolled. The animals were obtained from the Pasture Institute (Iran). The animals were housed in separate cages under standard temperature, humidity, and regular daylight cycle for one week.
The animals weighed 2-3 kg and were approximately the same age. The animals received an everyday nutritional regime and typically gained weight. In addition, a veterinarian ensured the systemic health of rabbits.
Through the surgery process, two implants were placed in the femur bone of each animal. One implant was treated by UV-C light; the other was not treated and was considered a control implant. The animals were divided into two groups and housed for two and four weeks. The animals were sacrificed at the end of the treatment period and the samples were subjected to microscopic analysis.

Surgery
The surgery was performed according to the protocol formerly provided by Gehrke et al. [19]. Furthermore, the experimental protocol was conducted according to the role and guidance provided by the Tehran University of Medical Science. For this purpose, twenty rabbits were obtained. The animals were slightly transferred

Histo-morphometric Analysis
For the histo-morphometric analysis, the prevalence of the newly formed bone was recorded. It calculated the specimens at the largest diameter of defects on a digital image (E450; Nikon, Japan) taken at 40× magnification using Iranian histo-morphometric analysis software (IHMA v1, Shaheed Beheshti University, Iran). The percent of cortical one formation and bone marrow formation was recorded (Figure 4).

Statistical Analysis
The sample size was calculated according to the previous study by Gehrke et al. [19] and below the mathe-

Results
The results of the normality test using the Kolmogorov-Smirnov test confirmed normal distribution between control and treatment groups in all time trials. Moreover, results of chi-square analysis showed that there was no significant difference between inflammation (p< 0.487), bleeding (p=1.0), peri-implant tissues (p=0.086).
In other words, exposure to UV-C had no significant effect on inflammation, bleeding, and tissues forming around the implant. However, results showed that irradi-

Effect of UV-C radiation of implants on cortical bone
ANOVA test results showed no significant difference for cortical bone formation in different time trials (p= 0.074, Table 1). However, the chi-square test showed that the percentage of cortical bone formation was significantly higher in the treatment group (p=0.001). More over, results showed that the interaction between groups and time was not significantly different ( Figure 5).

Effect of UV-C radiation of implants on cancellous bone
ANOVA test results showed no significant difference   Figure 6).

Effect of UV-C radiation of implants on new bone formation
ANOVA test results showed no significant difference for total new bone formation in different time trials (p= 0.098). The chi-square test showed that although the percentage of cancellous bone formation in two weeks was higher than in four weeks, this difference was not statistically significant (p= 0.811, Table 3).

Figure 6:
The diagram shows the interaction between the mean of cortical bone formation in treatment and control groups with different time trials However results showed that total new bone formation was significantly higher than the control group (p= 0.001). Moreover, results showed that the interaction between groups and time was not significantly different ( Figure 7).

Discussion
In this study, the histo-morphometric effect of UV-C irradiation on osseointegration of dental implants was evaluated. Results showed that new bone formation on the implant exposed to UV-C was significantly higher than in control groups. An increase in bone formation is affected by several factors, including (1)  The diagram shows the mean of total bone formation in treatment and control groups with different time trials prove in cytoskeletal structure and Vinculin expression [22]. It is noteworthy that the processes mentioned above are not separate phenomena; for instance, an increase in protein attachment increases the attachment of osteoblasts, and the growth of osteoblasts leads to cellular differentiation. These phenomena consequently lead to osteogenesis and osseointegration [10]. Evidence shows that the contamination of the titanium surface by saliva impaired the osteoblastic function of the titanium surface [20]. In other words, UV radiation clears biological pollutants and consequently changes the osteoblastic function of the titanium surface [20]. Furthermore, much evidence revealed that UV-C has antimicrobial potential [23][24][25]. The results of our study also suggest that UV-C could improve osseointegration.
Another evidence shows that UV radiation improves osteoblastic functions, such as alkaline phosphatase activity and calcium mineralization [21]. It was revealed that UV radiation changes the physicochemical properties of the titanium surface and significantly improves osteoblast attachment and function [21]. Moreover, results showed that a robust hydrophilic feature appears upon UV irradiation on the titanium surface [21]. The future reduces the water-droplet angle with the titanium surface to less than five degrees. Notably, the nonirradiated titanium surface is a hydrophobic and waterdroplet angle with the titanium surface to less than five degrees [21]. In this background, the results of our study suggest that improvement in osseo-integration would be due to the inducement of a hydrophilic feature on the titanium surface that increases initial osteoblast attachment to the titanium surface.
Furthermore, our study found that new bone formation significantly increased by UV-C irradiation on titanium dental implants after two and four weeks.
However, in both groups, no significant difference was observed in new bone formation between two and four weeks of the experiment. Similar evidence revealed that exposure to titanium implants with UV radiation for four weeks significantly increased bone regeneration [26]. However, the extension of that experiment to twelve weeks showed no significant difference in osseointegration in the treatment groups with the control group [26]. It is noteworthy that the impacts of UV irradiation during long-term treatment are affected by the type of cell, experiment length, intensity, UV wave-length, and implant surface texture [27][28]. Improved osseo-integration on UV irradiated implant for extending four months is due to increased mineralization between bone and implant, elevation in Aluminum concentration, and increased oxygen concentration on implant surface [26][27][28]. Another study revealed that exposure to a titanium implant for twelve minutes after twelve to twenty-four days significantly increased bone regeneration and osseointegration in rats [29].
Our study observed no significant bone regeneration between two and four weeks of treatment. It could be due to two reasons: (1) the physiology of bone regeneration in rabbits changes after two weeks. In which bone formation reduces and enters the remodeling phase [27][28][29], (2) the studied animal has a low amount of cortical bone, and the bone primarily contains bone marrow that resists new bone formation [30]. Furthermore, inflammation, bleeding, and fibrosis issues were evaluated in our study. However, the non-parametric method's histologic analysis revealed no significant difference between the control and treatment groups. That suggests UV radiation had no significant effect on inflammation and bleeding around the implant.

Conclusion
The study showed that a titanium implant irradiated with UV-C for four hours significantly improved osseointegration and new bone formation. However, prolonging the experiment from two to four weeks had no significant effect on bone formation. Therefore, the study suggests that UV-C radiation can increase the success rate of implant treatment.

Acknowledgments
The

Conflict of Interest
The authors acknowledge that there is no competing int-